Crystal structure of a bovine neurophysin II dipeptide complex at 2.8 A determined from the single-wavelength anomalous scattering signal of an incorporated iodine atom.

Abstract

The crystal structure of a dipeptide complex of bovine neurophysin H has been solved at 2.8 A resolutionsolely by using single-wavelength anomalous scattering data from a single iodinated derivative. The asymmetric unit is an elongated tetramer of dimensions 110 x 40 X 30 A, composed of two dimers related by pseudo twofold symmetry. Each monomer consists of two homologous layers, each with four antiparaflel 3-strands. The two regions are connected by a helix followed by a long loop. Monomer-monomer contacts involve antiparallel 13-sheet interactions, which form a dimer with two layers of eight P-strands. (10), one of which is isomorphous with the crystals of a porcine NP-I complex (11). These contained 4, 8, and 12 molecules per asymmetric unit, respectively, suggesting that the basic aggregates of the NP-dipeptide complex in the crystals are tetramers that can self-associate to higher oligomers (10). In view of the probable importance of NP self-association in packaging of the precursor in the NSG (1, 4) and evidence that NP complexes can be present in the NSG as the precipitated or crystalline state (12, 13), analysis of the crystal structure of these complexes offers an opportunity for providing detailed structural information not only on NP folding and protein-peptide interactions but also on how the complexes might be packaged in NSG.The crystal structure of a bovine NP-II-dipeptide complex"l reported here was determined using only the singlewavelength anomalous scattering (SAS) data from an iodinated derivative crystal. Iodine was chosen as a probe because of its relatively large anomalous scattering signal (Alf' = 6.8) and its ease of incorporation into the dipeptide.The resulting derivative crystals contained iodine atoms covalently linked to the Phe residues of the bound hormone analogs.Here we report the structure of a NP molecule, as well as aspects of the methodology used in solving the structure. METHODSCocrystaflization. Bovine NP-I was purified as described (14). The dipeptide para-iodo-L-phenylalanyltyrosine amide (I-Phe-Tyr-NH2) was custom-synthesized by Peninsula Laboratories and was demonstrated by using circular dichroism (8) to bind to the hormone-binding site with high affinity. Crystals of the NP-dipeptide complex were grown at pH 7.5 by using a modification of the procedure of Yoo et al. (9); crystals of the complex of the corresponding noniodinated peptide served as seeds. The crystals obtained were nearly isomorphous with those of the complex of the uniodinated peptide, having four molecules (chains) per asymmetric unit and space group P212121 with a = 120.0 A, b = 69.4 A, and c = 62.4 A.

Dominant pro-vasopressin mutants that cause diabetes insipidus form disulfide-linked fibrillar aggregates in the endoplasmic reticulum

“…For frameshift and point mutations affecting cysteine residues normally involved in disulfide bonds, misfolding is an obvious consequence. Failure to cleave the signal sequence has a more subtle effect, as it prevents the N-terminus of the vasopressin sequence from folding into NPII, a requirement for the stabilization of the disulfide bond within the hormone segment (Beuret et al, 1999;Chen et al, 1991). How can misfolding and ER retention cause cytotoxicity?…”

Molecular Biology of Hereditary Diabetes Insipidus

“…Additional posttranslation processing occurs within neurosecretory vesicles during transport of the precursor protein to axon terminals in the posterior pituitary, yielding AVP, neurophysin II, and the glycopeptide. The AVP-neurophysin II complex forms tetramers that can self-associate to form higher oligomers (11). Neurophysins should be seen as chaperone-like molecules facilitating intracellular transport in magnocellular cells.…”

Diabetes Insipidus: A Review

“…When an action potential causes an influx of Ca 2+ , the LDCVs fuse to the nerve terminal, releasing their contents via exocytosis. At normal plasma pH, NPII dissociates from ADH [7]. The LDCVs are then recovered via endocytosis and undergo retrograde transport to the cell body, where they are either reused, or degraded by lysosomes [5].…”